The long-term objective of this project is to understand, not only the structure and function of the vitamin K epoxide reductase (VKOR), but also the vitamin K cycle as a whole. Vitamin K-dependent carboxylation is an essential post-translational modification for proteins important in several physiologic functions, including blood coagulation and bone metabolism. VKOR is the target of warfarin, the most widely prescribed anti-coagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is still under-utilized because of fear of bleeding. It is hoped that research funded by this grant will lead to improved anti- thrombotic therapies that will avoid some of the problems related to warfarin treatment. Specifically, we propose to accomplish the following during the tenure of this grant: (1) using standard purification and enzyme assay methods, RNAi technologies, and modified expression cloning, we will investigate the vitamin K cycle to identify components other than the vitamin K epoxide reductase (VKOR) necessary for the production of vitamin K hydroquinone, (2) employing electron crystallography, biochemical and molecular biological techniques, conduct studies to elucidate the three- dimensional structure of VKOR, and (3) using both in vivo and in vitro methods investigate the structural characteristics necessary for VKOR. PUBLIC HEALTH RELEVANCE: In addition to development of new ways of controlling thromboses, knowledge gained from our research on the vitamin K cycle should give insight into the vitamin's apparently major role in bone formation. While the contribution to bone metabolism is clear, it is still controversial whether vitamin K is useful in treating or slowing progression of bone density loss in osteoporosis. Another developing area relevant to public health is the role of coagulation/thrombosis in cancer, and in turn whether vitamin K and its antagonists have a place in cancer treatment.